This application relates to the NIH efforts to develop and adapt biological assays for use in HTS. Our team has extensive experience in drug discovery and pharmaceutical screening campaigns. Our overall aim is to develop assays for HTS of small molecule compounds that display affinity to the neuropeptide Y (NPY) Y2- receptor. Specifically, we seek to identify compounds that are functional antagonists at the human Y2- receptor. Following optimization, also to be pursued at Scripps by individuals with much pharmaceutical industry experience, such a compound might be clinically useful in anxiety, depression and/or alcoholism. No clinically testable compounds that inhibit signaling of NPY through the Y2-receptor are in existence today. The NPY-Y2 receptor was first described by us previously and recent studies in human in particular have made us more convinced than ever that it is an important target in psychiatric disease and addiction. SPECIFIC OBJECTIVES AND AIMS: We will use a modified version of the HitHunter cAMP assay. This assay features high signal to background ratios for monitoring cellular activation of GPCRs, particularly G.i -coupled receptors. It is a homogeneous microtiter plate assay for measuring cellular cAMP from cell lysates using Enzyme Fragment Complementation (EFC). We aim to initially implement the DiscoveRx HitHunter cAMP XS technology and then develop further assays described herein, amenable to HTS (moving from 384- to 1536- well format) to screen a 600,000 compound library utilizing robotics for NPY-Y2 receptor antagonists. We will develop and use HA-Y2/CHO-K1 cells and 50% inhibition of NPY at 10 microM compound, measured at a single point will define hits. Confirmation of hits and potencies will be in 10 point dose response curves. Counter-screening of the NPY-Y1 receptor will help evaluate selectivity and parse down hits. IC50 determination will allow selection of compounds for chemical optimization. A radioactive detection method will be used to eliminate "false positives". Successful development and implementation of this assay format will pave the way for future G.s and G.i- coupled receptor HTS campaigns. RELEVANCE TO PUBLIC HEALTH: Many lines of evidence from studies on humans (and animals) suggest that the brain NPY-Y2 receptor is involved in affective disorders (depression as well as anxiety) and in alcoholism. Notably, only a few scattered efforts have previously focused on developing a Y2 receptor drug for psychiatric purposes. Our approach is novel, powerful and part of a comprehensive strategy to succeed in developing such a drug to alleviate human suffering.